Lipids are esters of long chain fatty acids (generally C14 to C24 saturated and unsaturated fatty acids in animal fats) and polyols such as glycerol, glycerol phosphates, alkyl glyceryl ethers, glycerol phosphoryl-choline, glycerol phosphoryl-serine, glycerol phosphoryl-ethanolamine, and the like. Lipids, in the form of cell membranes and fats, for example, constitute a significant proportion of animal body weight (e.g., about 5% to 25% of body weight in normal humans).
Lipids are not water-soluble, and generally do not cross biological membranes efficiently by simple diffusion. Dietary lipids are taken up primarily by hydrolysis of fatty acyl moieties from their corresponding polyol moiety and diffusion of the two moieties across the gut wall (although limited uptake of intact lipids occurs). Following absorption, lipids are reformed by reestablishment of ester bonds between polyol and fatty acyl moieties, and lipids are delivered throughout the body in esterified form (generally in lipoprotein-containing particles such as chylomicrons, very low, intermediate, low, and high density lipoprotein particles, and the like). Prior to uptake by cells (either for storage or for metabolism), lipids must again be hydrolyzed in order to facilitate passage across the cell membrane. Thus, enzymes which catalyze formation and hydrolysis of the ester bonds between fatty acyl moieties and polyol moieties of lipids must be present at several physiological locations, and the particular activities catalyzed by these enzymes (xe2x80x98lipasesxe2x80x99 ) varies depending on the physiological location and function of the enzyme.
A number of lipase enzymes have been characterized in various organisms, including in humans. However, it is far from clear that all physiologically relevant lipases have been discovered or characterized. The present invention provides novel nucleotide and amino acid sequence information corresponding to one or more human lipases.
The present invention is based, at least in part, on discovery of human cDNA molecules which encode lipase proteins such as the one herein designated MLip-1. These proteins catalyze formation and cleavage of ester bonds between fatty acyl moieties and glyceride moieties. MLip-1 protein, fragments thereof, derivatives thereof, and variants thereof are collectively referred to herein as polypeptides of the invention or proteins of the invention. Nucleic acid molecules encoding polypeptides of the invention (i.e., nucleic acids encoding MLip-1 protein, fragments thereof, derivatives thereof, and variants thereof) are collectively referred to as nucleic acids of the invention.
The nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes, particularly including processes which involve lipid metabolism and pancreatic function. Accordingly, in one aspect, the present invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable as primers or hybridization probes for detection of nucleic acids encoding a polypeptide of the invention.
The invention also includes nucleic acid molecules which are at least 40% (or, for example, 50%, 60%, 70%, 80%, 90%, 95%, or 98% or more) identical to the nucleotide sequence of either of SEQ ID NOs: 1 and 2, or a complement thereof.
The invention includes nucleic acid molecules which include a fragment of at least 56 (or, for example, 58, 60, 70, 80, 100, 125, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, or 2352) consecutive nucleotide residues of either of SEQ ID NOs: 1 and 2, or a complement thereof.
The invention also includes nucleic acid molecules which have a nucleotide sequence encoding a protein having an amino acid sequence that is at least 50% (or, for example, 60%, 70%, 80%, 90%, 95%, or 98% or more) identical to all or residues about 18-467 of the amino acid sequence SEQ ID NO: 3, or a complement thereof.
In certain embodiments, the nucleic acid molecules have the nucleotide sequence of either of SEQ ID NOs: 1 and 2.
Also within the invention are nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 3, the fragment including at least 17 (or, for example, 18, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, or 467) consecutive amino acid residues of SEQ ID NO: 3.
The invention includes nucleic acid molecules which encode a naturally-occurring allelic variant of a polypeptide having the amino acid sequence of SEQ ID NO: 3, wherein the nucleic acid molecule hybridizes under stringent conditions with a nucleic acid molecule having a nucleic acid sequence comprising either of SEQ ID NOs: 1 and 2, or a complement thereof.
The invention also includes nucleic acid molecules that hybridize under stringent conditions with a nucleic acid molecule having the nucleotide sequence of either of SEQ ID NOs: 1 and 2, or a complement thereof. In other embodiments, the nucleic acid molecules are at least 56 (or, for example, 58, 60, 70, 80, 100, 125, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, or 2352) nucleotides in length and hybridize under stringent conditions with a nucleic acid molecule having the nucleotide sequence of either of SEQ ID NOs: 1 and 2, or a complement thereof. In some embodiments, the isolated nucleic acid molecules encode an immature or mature form of a polypeptide of the invention. In other embodiments, the invention provides an isolated nucleic acid molecule which is antisense with respect to the coding strand of a nucleic acid of the invention.
Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. In a related aspect, the invention provides isolated host cells, e.g., mammalian and non-mammalian cells, containing such a vector or a nucleic acid of the invention. The invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector encoding a polypeptide of the invention such that the polypeptide of the invention is produced.
Another aspect of this invention includes isolated or recombinant proteins and polypeptides of the invention. Isolated polypeptides or proteins have an amino acid sequence that is at least about 50% (or, for example, 60%, 75%, 90%, 95%, or 98% or more) identical to all or a portion of the amino acid sequence of SEQ ID NO: 3. Exemplary polypeptides of the invention include a polypeptide having the amino acid sequence SEQ ID NO: 3, a polypeptide having the amino acid sequence of only residues 1 to about 17 of SEQ ID NO: 3 (i.e., the signal peptide of MLip-1), a polypeptide having the amino acid sequence of about residues 18 to 467 of SEQ ID NO: 3 (i.e., mature MLip-1 protein), and a polypeptide corresponding to a solvent-exposed portion of MLip-1 protein (e.g., about amino acid residues 80 to 105 of SEQ ID NO: 3).
Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 40% (or, for example, 50%, 75%, 85%, or 95% or more) identical to the nucleic acid sequence of either of SEQ ID NOs: 1 and 2, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions with a nucleic acid molecule having the nucleotide sequence of either of SEQ ID NOs: 1 and 2.
Also within the invention are polypeptides which are naturally-occurring allelic variants of a polypeptide that has the amino acid sequence SEQ ID NO: 3, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes under stringent conditions with a nucleic acid molecule having the nucleotide sequence of either of SEQ ID NOs: 1 and 2, or a complement thereof.
In certain embodiments, proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide. An activity or a biological activity of a polypeptide of the invention refers to an activity exerted by the polypeptide of the invention on a responsive cell, on a portion of a cell (e.g., a cell membrane), on a cellular nutrient (e.g., a triglyceride or other lipid), or on a cellular metabolite or other product (e.g., cholesterol or membrane lipids). Such activity can be assessed in vivo or in vitro, according to standard techniques. MLip-1 polypeptides of the invention exhibit lipase activity, and can be involved in a number of bodily functions including, for example, dietary fat degradation and absorption, cholesterol biosynthesis, and maintenance of plasma lipid and lipoprotein levels. Such activities can, for example, be an enzymatic activity exerted by a polypeptide of the invention on another protein or on a non-protein substrate (e.g., on a lipoprotein particle or a triglyceride).
By way of example, protein MLip-1, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with MLip-1 (collectively xe2x80x9cMLip-1-related moleculesxe2x80x9d) exhibit the ability to affect growth, proliferation, survival, differentiation, and activity of pancreatic tissue, in which MLip-1 is expressed. MLip-1-related molecules can be used to prevent, diagnose, or treat disorders relating to inappropriate lipid metabolism and aberrant pancreatic function. Exemplary disorders for which MLip-1-related molecules are useful include diabetes, obesity, nutritional disorders (e.g., lipid malabsorption and malnutrition), metabolic disorders (particularly including lipid metabolism anomalies such as hyperlipidemia of types I to V and hypolipidemia), pancreatitis, obstruction of the pancreatic duct, various lipidoses (e.g., Gaucher""s disease and Niemann-Pick disease), atherosclerosis, arteriosclerosis, coronary artery disease, perforated peptic ulcer, abdominal lesions, intestinal obstruction, peritonitis, and other diseases and disorders associated with aberrant or physiologically inappropriate lipase and lipase-like activity.
In one embodiment, a polypeptide of the invention has an amino acid sequence that is sufficiently identical to an identified domain of MLip-1 (e.g., a domain present at the surface of MLip-1 or the lipase domain described herein) that the polypeptide exhibits an antigenic or enzymatic characteristic of MLip-1. Such polypeptides comprise at least about 17 (18, 20, 25, 35, 50, 75, 100, 150, 200, 250, or 300 or more) amino acid residues, of which at least about 65%, preferably at least about 75%, and more preferably at least about 85%, 95%, or 98% are identical or similar (representing conservative amino acid substitutions; i.e., between amino acids having similar side chain moieties). Exemplary antigenic and enzymatic characteristics of MLip-1 which are exhibited by such polypeptides include lipase activity, ability to bind with molecules (e.g., enzymatic substrates or cell-surface or lipoprotein particle surface sites) with which MLip-1 is able to bind, and ability to induce production of antibody substances (e.g., free and cell-surface-bound immunoglobulins such as antibodies and T cell receptors) which bind specifically with an epitope which occurs at or near the surface of MLip-1 protein.
The polypeptides of the present invention, or biologically active portions thereof, can be operably linked with a heterologous amino acid sequence to form fusion proteins. In addition, one or more polypeptides of the invention or biologically active portions thereof can be incorporated into pharmaceutical compositions, which can optionally include pharmaceutically acceptable carriers. Such pharmaceutical compositions can be used to treat or prevent one or more of the disorders identified herein.
The invention encompasses antibody substances that specifically bind with a polypeptide of the invention including, for example, MLip-1 protein and fragments thereof. Exemplary antibody substances that are included within the scope of the invention are monoclonal and polyclonal antibodies, antibody fragments, single-chain antibodies, free and cell-surface-bound antibodies, and T cell receptors. These antibody substances can be made, for example, by providing the polypeptide of the invention to an immunocompetent vertebrate and thereafter harvesting blood or serum from the vertebrate. Antibody substances can, alternatively, be generated by screening a library of phage (e.g., a filamentous phage such as M13) which express one or more immunoglobulin subunits (e.g., IgG heavy chains) on their surface to identify phage particles which display a subunit which binds with MLip-1 or an epitope thereof.
In another aspect, the present invention provides methods for detecting activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting such activity (e.g., a labeled substrate or another compound that can be detected after being acted upon by an active polypeptide of the invention), with an agent which binds specifically with a polypeptide of the invention (e.g., an antibody substance of the invention), or with an agent for detecting production of an RNA encoding a polypeptide of the invention (e.g., a reverse transcriptase primer complementary to a portion of an mRNA encoding the polypeptide).
The present invention also provides diagnostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a polypeptide of the invention; (ii) mis-regulation of a gene encoding a polypeptide of the invention; and (iii) aberrant post-translational modification of a polypeptide of the invention wherein a wild-type form of the gene encodes a polypeptide which exhibits at least one activity of the polypeptide of the invention. Such diagnostic assays include, for example, (i) comparing the nucleotide sequence of all or part of a gene which encodes a polypeptide of the invention and which is obtained from a subject with the nucleotide sequence (or the corresponding part thereof) of a gene obtained from a subject having a non-mutated MLip-1 gene or one of SEQ ID NOs: 1 and 2; (ii) comparing the presence or level in a sample obtained from a subject of a polypeptide or polynucleotide corresponding to all or part of MLip-1 with the presence or level in other samples (preferably samples of the same type) obtained from one or more other subjects; and (iii) determining whether a polypeptide or polynucleotide corresponding to all or a part of MLip-1 that includes a sequence corresponding to a post-translational modification site identified herein, or determining whether a polypeptide of the invention is modified at such a site.
In another aspect, the invention provides a method for identifying a compound that modulates (i.e., inhibits or enhances) the activity of or binds with a polypeptide of the invention. In general, such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide. Such methods can be performed in vitro or in vivo (e.g., in an animal which naturally expresses the polypeptide or nucleic acid or in an animal that has been modified such that it artificially expresses the polypeptide or nucleic acid).
The invention also includes methods of identifying a compound that modulates expression of a polypeptide or nucleic acid of the invention by measuring expression of the polypeptide or nucleic acid in the presence and absence of the compound.
In another aspect, the invention provides methods for modulating activity of a polypeptide of the invention, the methods comprising contacting a cell with an agent that modulates the activity or expression of the polypeptide, such that activity or expression in the cell is modulated (e.g., by contacting the cell with a sufficient amount of the agent). In one embodiment, the agent is an antibody that specifically binds with a polypeptide of the invention. In another embodiment, the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an RNA (e.g., a pre-mRNA or an mRNA) encoding the polypeptide of the invention. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense with respect to the coding strand of an RNA encoding a polypeptide of the invention. In still other embodiments, the agent is a small molecule (e.g., a compound having a molecular weight less than about 5,000) which modulates activity or expression of a polypeptide or nucleic acid of the invention.
In yet another aspect, the invention includes a method of treating a patient afflicted with a disorder characterized by aberrant activity of a polypeptide of the invention, or by aberrant expression of a nucleic acid of the invention. The method comprises administering to the patient an agent (e.g., a nucleic acid, polypeptide, small molecule, antibody, or the like) in an amount effective to modulate the activity of the polypeptide in the patient or a to modulate the expression of the nucleic acid in the patient. Following administration of the agent, at least one symptom of the disorder is alleviated. In an alternative method of treating a patient afflicted with a disorder associated with aberrant activity or expression of MLip-1 protein, the method comprises administering to the patient, in an amount effective to modulate the level of activity of the protein in the patient, an agent selected from the group consisting of
i) a polypeptide of the invention;
ii) a variant of a polypeptide of the invention;
iii) a nucleic acid encoding a polypeptide of the invention; and
iv) an antisense nucleic acid which is capable of annealing with either of an mRNA encoding a polypeptide of the invention and a portion of a genomic DNA encoding a polypeptide of the invention.
Following administration of the agent, at least one symptom of the disorder is alleviated.
In still another aspect, the invention relates to a method of diagnosing a disorder associated with aberrant expression of MLip-1 protein in a patient. This method comprises assessing the level of expression of the gene encoding the protein (e.g., by assessing the quantity of a corresponding RNA, the quantity of a corresponding protein, or the activity of a corresponding protein) in the patient and comparing the level of expression of the gene with the normal level of expression of the gene in a human not afflicted with the disorder. A difference between the level of expression of the gene in the patient and the normal level is an indication that the patient is afflicted with the disorder.
The invention also includes a method of diagnosing a disorder associated with expression of an aberrant or mutated MLip-1 protein in a patient. This method can be performed by comparing the nucleotide sequence of a nucleic acid encoding MLip-1 protein in a patient with a nucleotide sequence (e.g., one of SEQ ID NOs: 1 and 2) encoding MLip-1 protein in a subject not afflicted with the disorder. A difference between the two nucleotide sequences is an indication that the patient is afflicted with the disorder. This method can also be performed by comparing the amino acid sequence of a portion (i.e., including all) of MLip-1 protein in a sample obtained from the patient with the amino acid sequence of the same portion of MLip-1 protein in a sample obtained from a subject not afflicted with the disorder. A difference between the two amino acid sequences is an indication that the patient is afflicted with the disorder.
In yet another aspect, the invention relates to a method of determining whether a patient is likely to become afflicted in the future with a disorder associated with aberrant expression of MLip-1 protein or with expression of an aberrant or mutated MLip-1 protein. In various embodiments, these prognostic methods comprise (i) comparing the nucleotide sequence of a nucleic acid encoding MLip-1 protein in a sample obtained from a patient with a nucleotide sequence (e.g., one of SEQ ID NOs: 1 and 2) encoding MLip-1 protein in a subject known not to be afflicted; and not to be predisposed to becoming afflicted with the disorder or (ii) comparing the amino acid sequence of all or a portion of MLip-1 protein obtained from a patient with the amino acid sequence (e.g., SEQ ID NO: 3) of MLip-1 protein obtained from a non-afflicted subject.
Other features and advantages of the invention will be apparent from the following detailed description and claims.